During sporulation the bacterium, Bacillus thuringiensis (Bt) produces a parasporal inclusion with selective toxicity to insect larvae. Generally, the inclusion is deposited outside the exosporium and released into the environment separately from the spore. In subspecies finitimus however, two parasporal inclusions are produced. A minor inclusion forms outside the exosporium and is released into the environment independently of the spore. The second, major inclusion and the spore share the common exosporium and ar released together. The physiological significance of an enclosed inclusion and the mechanism for targeting parasporal inclusion proteins within the exosporium are not known and are the bases of the proposed study. The long term objective of the proposed project is to increase understanding of Bt toxin expression and targeting in the cell with the ultimate goal of characterizing genes that may be potentially important in designing new pesticidal agents and devising strategies for their use. Specifically the project is designed to identify factors important in exosporium enclosure of inclusions and to determine the contribution of the exosporium to distribution, stability, and toxicity of inclusions. Experiments will be designed to clone the gene for enclosed parasporal inclusion protein of subspecies finitimus; to use the cloned gene as a tool to identify factors responsible for exosporium enclosure of inclusions; to enclose an inclusion within an exosporium artificially; and to determine the effects of the exosporium on toxicity, stability, and distribution of inclusions.